Automated image analysis method for detecting and quantifying macrovesicular steatosis in hematoxylin and eosin-stained histology images of human livers.

Large-droplet macrovesicular steatosis (ld-MaS) in more than 30% of liver graft hepatocytes is a major risk factor for liver transplantation. An accurate assessment of the ld-MaS percentage is crucial for determining liver graft transplantability, which is currently based on pathologists' evaluations of hematoxylin and eosin (H&E)-stained liver histology specimens, with the predominant criteria being the relative size of the lipid droplets (LDs) and their propensity to displace a hepatocyte's nucleus to the cell periphery. Automated image analysis systems aimed at objectively and reproducibly quantifying ld-MaS do not accurately differentiate large LDs from small-droplet macrovesicular steatosis and do not take into account LD-mediated nuclear displacement; this leads to a poor correlation with pathologists' assessments.

Rat hepatocyte culture model of macrosteatosis: effect of macrosteatosis induction and reversal on viability and liver-specific function.

Background & Aims: A common cause of liver donor ineligibility is macrosteatosis. Recovery of such livers could enhance donor availability. Living donor studies have shown diet-induced reduction of macrosteatosis enables transplantation.

Is extracorporeal hypothermic machine perfusion of the liver better than the 'good old icebox'?

Purpose Of Review: To underscore the advantages of hypothermic machine perfusion (HMP) in light of the recent discoveries.

Recent Findings: The phase I clinical trial of liver HMP has suggested superiority over static cold storage (SCS) preservation. The liver transplant arena has gained more sophisticated insights into the molecular mechanism and biomechanical understanding behind the benefits of HMP.

Hepatic cell-to-cell transmission of small silencing RNA can extend the therapeutic reach of RNA interference (RNAi).

Background/aims: RNA interference (RNAi), a sequence-specific gene silencing technology triggered by small interfering RNA (siRNA), represents promising new avenues for treatment of various liver diseases including hepatitis C virus (HCV) infection. In plants and invertebrates, RNAi provides an important mechanism of cellular defence against viral pathogens and is dependent on the spread of siRNA to neighbouring cells. A study was undertaken to investigate whether vector-delivered RNAi can transfer between hepatic cells in vitro and in mice, and whether this exchange could extend the therapeutic effect of RNAi against HCV infection.

Protective effects of hypothermic ex vivo perfusion on ischemia/reperfusion injury and transplant outcomes.

Hypothermic machine preservation (HMP) has been used in renal transplantation since the late 1960s with recent robust prospective, multicenter data showing lower rates of delayed graft function and improved graft survival. Although now clearly beneficial for renal transplantation, extrarenal machine perfusion has remained predominantly in preclinical investigations. Pancreatic HMP has drawn little clinical interest because HMP has been suggested to cause graft edema and congestion, which is associated with early venous thrombosis and graft failure.

Molecular expression of acute phase mediators is attenuated by machine preservation in human liver transplantation: preliminary analysis of effluent, serum, and liver biopsies.

Background: Hypothermic machine perfusion (HMP) mitigates the effects of ischemia/reperfusion injury (IRI) in renal transplantation and preclinical work with livers. In liver transplantation, IRI increases the likelihood of primary graft dysfunction and is associated with significant morbidity. We recently completely the first phase 1 clinical trial of liver HMP at our center, and demonstrated improved clinical parameters and shorter duration of stay for patients who received grafts stored by HMP than patients who received grafts preserved in cold storage.

Production of multicopy shRNA lentiviral vectors for antiviral therapy.

For effective RNA interference (RNAi)-based therapies against viral infection, particularly highly mutational viruses like HCV and HIV, combinational strategies that target multiple regions within a viral genome are required to prevent resistance. The use of lentiviral vectors for combinatorial RNAi (coRNAi) offers possibilities to deliver multiple short hairpin RNA (shRNA) sequences simultaneously to individual cells while maintaining high expression levels required to suppress viral replication. By applying coRNAi, one can impart either a protective strategy, i.

Hypothermic machine preservation attenuates ischemia/reperfusion markers after liver transplantation: preliminary results.

Background: Hypothermic machine perfusion (HMP) has shown significant benefits in renal transplantation but is still in its infancy in liver transplantation. Potential benefits include diminished preservation injury and improved early graft function.

Methods: We analyzed liver tissue and effluent collected during our Phase 1 trial of liver HMP.

Combined antiviral activity of interferon-alpha and RNA interference directed against hepatitis C without affecting vector delivery and gene silencing.

The current standard interferon-alpha (IFN-alpha)-based therapy for chronic hepatitis C virus (HCV) infection is only effective in approximately half of the patients, prompting the need for alternative treatments. RNA interference (RNAi) represents novel approach to combat HCV by sequence-specific targeting of viral or host factors involved in infection. Monotherapy of RNAi, however, may lead to therapeutic resistance by mutational escape of the virus.

Hydroxyethyl starch-based preservation solutions enhance gene therapy vector delivery under hypothermic conditions.

Isolated liver perfusion offers a unique prospect for safe, effective targeting of gene therapies that can be directed against allograft rejection or recurrent diseases such as reinfection by hepatitis C virus (HCV). We aimed to examine the effect of organ preservation solutions on vector-based gene therapy delivery under hypothermic conditions. University of Wisconsin (UW) solution, histidine tryptophan ketoglutarate (HTK), EloHaes, sodium-poly(ethylene glycol)-UW solution [Institut Georges Lopez 1 solution (IGL-1)], and Dulbecco's modified Eagle's medium (DMEM) culture medium (control) were tested at 2 degrees C or 37 degrees C for lentiviral vector transduction efficiencies to the hepatoma cell line Huh-7 and primary human or mouse hepatocytes.

Impact of steroids on hepatitis C virus replication in vivo and in vitro.

Chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation. Transplantation outcome is often compromised by a rapid re-infection of the graft. Several factors have been implicated in the increased severity of recurrence, including steroid-based immunosuppression.

New therapeutic opportunities for hepatitis C based on small RNA.

Hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease, including cirrhosis and liver cancer and is therefore, the most common indication for liver transplantation. Conventional antiviral drugs such as pegylated interferon-alpha, taken in combination with ribavirin, represent a milestone in the therapy of this disease. However, due to different viral and host factors, clinical success can be achieved only in approximately half of patients, making urgent the requirement of exploiting alternative approaches for HCV therapy.

Mycophenolic acid inhibits hepatitis C virus replication and acts in synergy with cyclosporin A and interferon-alpha.

Background & Aims: Chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation. Clinical evidence suggests that particular immunosuppressive agents can have an influence on HCV recurrence. Cyclosporine A (CsA) specifically inhibits HCV replication through blocking the viral RNA polymerase enzyme NS5B.

Simultaneous targeting of HCV replication and viral binding with a single lentiviral vector containing multiple RNA interference expression cassettes.

Chronic hepatitis C virus (HCV) infection has a major medical impact and current treatments are often unsuccessful. RNA interference represents a promising new approach to tackling this problem. The current study details the design and testing of self-inactivating lentiviral vectors (LV) delivering RNA interference to prevent HCV replication and infection.

Human immunodeficiency virus type 1 Nef protein mediates neural cell death: a neurotoxic role for IP-10.

HIV-1 Nef is expressed in astrocytes, but a contribution to neuropathogenesis and the development of HIV-associated dementia (HAD) remains uncertain. To determine the neuropathogenic actions of the HIV-1 Nef protein, the brain-derived (YU-2) and blood-derived (NL4-3) Nef proteins were expressed in neural cells using an alphavirus vector, which resulted in astrocyte death (P < 0.001).

A1 adenosine receptor upregulation and activation attenuates neuroinflammation and demyelination in a model of multiple sclerosis.

The neuromodulator adenosine regulates immune activation and neuronal survival through specific G-protein-coupled receptors expressed on macrophages and neurons, including the A1 adenosine receptor (A1AR). Here we show that A1AR null (A1AR-/-) mice developed a severe progressive-relapsing form of experimental allergic encephalomyelitis (EAE) compared with their wild-type (A1AR+/+) littermates. Worsened demyelination, axonal injury, and enhanced activation of microglia/macrophages were observed in A1AR-/- animals.

Intracerebral hemorrhage induces macrophage activation and matrix metalloproteinases.

Intracerebral hemorrhage (ICH) is characterized by parenchymal hematoma formation with surrounding inflammation. Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of neurological diseases defined by inflammation and cell death. To investigate the expression profile and pathogenic aspects of MMPs in ICH, we examined MMP expression in vivo using a collagenase-induced rat model of ICH.

Up-regulation of proteinase-activated receptor 1 expression in astrocytes during HIV encephalitis.

Proteinase-activated receptor 1 (PAR-1) is a G protein-coupled receptor that is activated by thrombin and is implicated in the pathogenesis of inflammation. Although PAR-1 is expressed on immunocompetent cells within the brain such as astrocytes, little is known about its role in the pathogenesis of inflammatory brain diseases. Herein, we investigated PAR-1 regulation of brain inflammation by stimulating human astrocytic cells with thrombin or the selective PAR-1-activating peptide.